Method for detecting human kininogen using monoclonal antibodies thereto

ABSTRACT

Two novel cell lines, ATCC #HB-8963 and ATCC #HB-8964 produce monoclonal antibody to human kininogen. One of the antibodies specifically recognizes the heavy chain of high and low molecular weight kininogen (the later protein is identical to alpha cysteine protease inhibitor). The other antibody recognizes the light chain of high molecular weight kininogen. The hybridomas are formed by fusing spleen cells from immunized BALB/c AnSkh mice with P3X63Ag8 or SP2/0-Ag14 myeloma cells. Diagnostic, therapeutic and biochemical uses of the monoclonal antibodies are provided.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported by National Institutes ofHealth grants HL00694 and HL24365. The United States Government has aroyalty-free, nonexclusive and irrevocable license to practice theinvention.

This is a divisional of application Ser. No. 06/821,367, filed Jan. 26,1986, now U.S. Pat. No. 4,908,431.

FIELD OF THE INVENTION

This invention relates to hybrid cell lines for production of monoclonalantibodies to human kininogens, proteins which are found in nearly allhuman blood plasma, platelets, and kidney tissue. The invention alsorelates to the antibody produced by these cell lines, and to diagnostic,therapeutic and biochemical methods and compositions using the same.

BACKGROUND OF THE INVENTION

The plasma kininogens are single-chain glycoproteins which are presentin human blood plasma and tissues in two forms: high molecular weightkininogen (120 kDa) and low molecular weight kininogen (64 kDa). Thegene that controls the synthesis of both kininogens is the same(Kitamura, N. et al, Nature 305, 545, 1983). The difference between thehigh molecular weight form and the low molecular weight form is theaddition of an unique light chain by a posttranscriptional modificationon each of the molecules. The presence of a 56 kDa light chain on highmolecular weight kininogen gives this form of kininogen unique antigenicas well as functional properties. The plasma concentration of highmolecular weight kininogen is 0.67 micromolar, while the concentrationof low molecular weight kininogen is 2.4 micromolar.

The functions of the plasma kininogens are as follows. Both highmolecular weight and low molecular weight kininogen are parent moleculesfor the decapeptide, bradykinin, the most potent naturally-occurringvasodilitory substance. Bradykinin is best liberated from high molecularweight kininogen by plasma kallikrein, but low molecular weightkininogen is a better substrate for tissue kallikreins to releasebradykinin.

High molecular weight kininogen also functions as a cofactor for theactivation of the following plasma zymogens: factor XII, prekallikrein,and factor XI. These three plasma zymogens/enzymes, along with highmolecular weight kininogen, comprise the proteins of the contact phaseof plasma proteolysis. In addition to being a cofactor for activation ofeach of these plasma zymogens, high molecular weight kininogen is also asubstrate of each of their proteolytic forms. Moreover, high molecularweight kininogen serves to order the activation and inhibition of theseproteases since 75% of plasma prekallikrein and 90% of plasma factor XIcirculate in plasma in complex with high molecular weight kininogen. Theproteins of the contact phase of plasma proteolysis are a group ofproteins that link the plasma proteolytic systems of coagulation,fibrinolysis, complement activation, and blood pressure control.

Low molecular weight kininogen, the preferred substrate for the tissuekallikreins, has recently been identified as being identical to alphacysteine protease inhibitor--a major plasma inhibitor of cysteineproteases (Ohkubo, I. et al, Biochem. 23, 3891, 1984). Since the heavychain of both high molecular weight and low molecular weight kininogenare identical, high molecular weight kininogen has also been shown toinhibit cysteine proteases (Muller-Esterl, W., FEBS 182, 310, 1985;Sueyoshi, T. et al, FEBS 182, 193, 1985). High molecular weightkininogen has been shown in our laboratories to be the most potentinhibitor of the calcium-activated cysteine protease from platelets(Schmaier, A. H. et al, Blood 66 (Suppl):313A, 1985).

Using high molecular weight kininogen as an example, the molecularstructure of the kininogen molecules, as the structure relates to thefunction of the molecule, can be described. When the intact 120 kDa highmolecular weight kininogen molecule is cleaved by plasma kallikrein, theinitial two cleavages results in the liberation of the 1000 molecularweight peptide bradykinin, leaving a residual twochain molecule joinedtogether by disulfide bonds. A residual heavy chain (64 kDa) isidentical to the heavy chain of plasma low molecular weight kininogen(alpha cysteine protease inhibitor) which can function as a cysteineprotease inhibitor. Likewise, an intermediate 56 kDa light chain isformed which contains the unique antigenic and functional properties ofthe high molecular weight kininogen molecule. This light chain serves asthe site for the surface-mediated activities of the molecule that areinvolved in activating and regulating inhibition of the contact phasezymogens:factor XII, prekallikrein and factor XI. Further cleavage ofthe 56 kDa light chain of high molecular weight kininogen by kallikreinresults in a stable 46 kDa light chain with similar functionalproperties.

It is hypothesized, but not yet conclusively shown, that the role ofplasma kininogens as cysteine protease inhibitors limits the extent oftissue injury upon cytolytic destruction of cells by cysteine proteases.This type of injury occurs as result of complement activation andmechanical destruction. Purified plasma kininogen could be used as atherapeutic tool in the treatment of such conditions as peridontaldisease and intervertebral disc disease, where cysteine proteases arepathogenic in these disorders.

The surface-mediated activation of the proteins of the contact phase ofplasma proteolysis (factors XI and XII; prekallikrein; high molecularweight kininogen) occurs in pathologic states. Gram negative sepsis,typhoid fever, and acute attacks of hereditary angioedema are seriousdisease states wherein these proteins are altered and the contactactivation system is activated. Early detection of activation of thecontact system proteins could have prognostic and therapeutic results onthe outcome of treating the above conditions. The ability to developmeans for early detection of activation of the contact system proteinscan be developed by use of antigenic assays to activation fragments ofthese proteins.

Kohler and Milstein (Nature 256, 493-497, 1975) were the first todescribe the fusion of myeloma cells to immune spleen cells from mice togenerate continuous cell lines. These hybrid cell lines, or hybridomas,have characteristics that neither the parental myeloma cells norparental immune spleen cell possess. Hybridomas are capable ofcontinuously producing homogeneous (monoclonal) antibodies. Prior to thework of Kohler and Milstein, only polyclonal antisera could be obtained.

Although techniques for the production of hybridomas are now extensivelydescribed in the literature, e.g., Monoclonal Antibodies, Hybridomas: ANew Dimension In Biological Analysis, R. H. Kennet, T. J. McKearn, andK. B. Bechtol, eds., Plenum Press, New York and London (1980), there isno general method for obtaining successful monoclonal antibody-producinghybridomas which can be used with all antigens. Fusion techniques mustbe varied in each case to obtain hybridomas producing monoclonalantibody to the desired antigen. In order to obtain antibodies specificto a single antigen, laborious purification techniques are required toprovide highly purified antigen for immunization. The production ofmonoclonal antibodies for any given antigen is still a highly empiricalprocess.

The kininogens have been detected in blood by means of coagulant andimmunochemical assays using polyclonal antisera. There have been noreports of monoclonal antibodies against kininogens prior to the presentinvention. The dearth of literature accounts of monoclonal antibodies tothe kininogens is no doubt due to difficulties in the purification ofantigen and/or the lack of success in preparation of suitablehybridomas.

Hereinafter, "human kininogen", shall mean, unless otherwise indicated,both the high and low molecular weight forms of any kininogen molecule,in all its various forms derived from human plasma, platelets, kidney,skin, leukocytes or other tissues or organs, regardless of whether foundin the fluid or the tissue phase.

"HMWK" shall mean high molecular weight human kininogen.

"LMWK" shall mean low molecular weight human kininogen, also known asalpha cysteine protease inhibitor, or alphalthiol protease inhibitor, oralpha₂ -thiol protease inhibitor.

"Heavy chain" shall mean, when referring or relating to human kininogen,the 64 kDa kallikrein-cleavage fragment of HMWK, said fragment beingidentical to the heavy chain of LMWK.

"Light chain" shall mean, when referring or relating to human kininogen,the 56 kDa intermediate kallikreincleavage fragment of HMWK which hasthe ability to correct the coagulant defect in total kininogen-deficientplasma.

SUMMARY OF THE INVENTION

According to the present invention, novel hybridomas have been preparedproviding cell lines producing monoclonal antibodies which specificallybind to an antigenic determinant of human kininogen. Each hybridomacomprises a cell hybrid formed by fusion of cells from a myeloma lineand spleen cells from a donor previously immunized with human kininogen.The hybridomas are, respectively, ATCC #HB-8963 and #HB-8964. Eachantibody so produced is specific for an antigenic determinant of humankininogen. The purified monoclonal antibody contains essentially noother anti-human immunoglobulin. The hybridomas may be cultured in vitroto secrete antibodies.

The hybrid cell lines of the present invention may be prepared by firstimmunizing mice with purified human kininogen. The spleen cells are thenremoved and a suspension thereof is made. The spleen cells are fusedwith mouse myeloma cells in the presence of a fusion promotor. The fusedcells are diluted and cultured in separate wells in a medium which willnot support the unfused myeloma cells. The supernatant in each well isassayed for the presence of antibody to human kininogen by anenzyme-linked immunosorbent assay (ELISA). Hybridomas secreting antibodywhich binds to human kininogen are selected and cloned.

The hybridomas are cultured in a suitable medium and the antibody isrecovered from the supernatant. Alternatively, the clones aretransferred intraperitoneally into mice, and the resulting malignantascites and serum containing the desired antibody are harvested.

A method for detecting the level of human kininogen in specimens ofinterest is provided. A monoclonal antibody according to the presentinvention, which binds to an antigenic determinant of human kininogen,is contacted with the specimen under conditions such that an immunecomplex forms between the monoclonal antibody and any kininogen in thespecimen. The material bound by the antibody is measured by standardassay means. The invention is particularly useful for detectingvariations in the level of plasma kininogens in human plasma.

The antibodies of the invention may be bound to an immobilized matrixand used to purify either HMWK or LMWK. Matrices of this type chargedwith HMWK may be used to purify prekallikrein and factor XI, which bindto HMWK.

The subject hybridomas are identified herein by the same number assignedto the antibody produced thereby. Thus, for example, the designation"2B5" pertains to both the hybridoma 2B5-G4-F4 and the monoclonalantibody produced by this hybridoma. The particular material referredto, that is, hybridoma versus antibody, is apparent from the context.

The subject hybridomas were deposited in the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, and were giventhe following ATCC accession numbers: #HB-8963 for 2B5-G4-F4; #HB-8964for CllCl. The hybridomas were deposited on Dec. 6, 1985.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of the neutralization of HMWK coagulant activity by the2B5 and CllCl antibodies. Percent inhibition of HMWK coagulant activity(ordinate) is plotted against antibody dilution (abscissa). The (∘)curve represents inhibition produced by the CllCl antibody; the ○ curveis inhibition by the 2B5 antibody.

FIG. 2 is a plot of the competition inhibition of the 2B5 antibodyproduced by normal plasma (∘ . . . ∘), HMWK deficient plasma (□ . . .□), and total kininogen deficient plasma (∘ . . .∘).

FIG. 3 is an immunoblot generated with the 2B5 antibody from thealkaline polyacrylamide gel electrophoresis of (from left to right) (i)kininogen antigen in normal plasma ("NHP"), (ii) kininogen antigen inHMWK-deficient plasma ("FITZ PLASMA"), (iii) purified HMWK ("HMWK") and(iv) purified HMWK detected by Coomasie blue staining ("HMWK COOMASIE").

FIG. 4 is a plot of a four-parameter logistic function calculation ofthe competition inhibition of the 2B5 antibody by normal plasma.

FIG. 5 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) of purified, cleaved HMWK chains after alkylation andreduction. The lane marked "HMWK" represents purified HMWK. The lanemarked "BOUND" represents HMWK heavy chain that is bound to an affinitycolumn made from coupling the 2B5 antibody to cyanogen bromide-activatedSepharose 4B. The lane marked "UNBOUND" represents two forms of HMWKlight chain that did not bind to the antibody attached to the supportmatrix.

FIG. 6 is a plot of the competition inhibition of the CllCl antibody bykininogen antigen in normal plasma (NHP) and HMWK deficient plasma(FITZ).

FIG. 7 is an immunoblot generated with the CllCl antibody from thealkaline polyacrylamide gel electrophoresis of (from left to right) (i)kininogen antigen from normal plasma ("NHP"), (ii) kininogen antigenfrom HMWK-deficient plasma ("FITZ"), (iii) purified HMWK ("HMWK"), and(iv) purified HMWK detected by staining with Coomasie blue ("HMWK(stained)").

FIG. 8 is a plot of the four-parameter logistic function calculation ofthe competition inhibition of the CllCl antibody by normal plasma.

FIG. 9 is a SDS-PAGE immunoblot using the CllCl antibody. The lanemarked "HMWK" represents purified HMWK immunoprecipitated with the CllClantibody. The "PLT LYS" lane indicates the immunoorecipitation ofplatelet high molecular weight kininogen with CllCl antibody. "HMWK PLTLYS" represents purified ¹²⁵ I-HMWK placed into a platelet lysate oftotal kininogen-deficient platelets and immunoprecipitated with theCllCl antibody. The "CN" lane represents a control immunoprecipitationfrom a platelet lysate using the CllCl antibody without the addition ofProtein A.

DETAILED DESCRIPTION OF THE INVENTION

The cell hybrids of the present invention produce monoclonal antibodiesthat react with human plasma kininogen. The monoclonal antibody 2B5reacts with kininogen antigen in normal plasma with two bands, kininogenantigen in HMWK deficient plasma, and purified alkylated and reducedHMWK heavy chain. In addition, 2B5 does not neutralize HMWK coagulantactivity in plasma. These combined studies indicate that the 2B5monoclonal antibody is directed to the HMWK heavy chain, which is thesame as the heavy chain of LMWK. The monoclonal antibody CllCl reactswith kininogen antigen (one band) in normal plasma but not withkininogen antigen in HMWK deficient plasma. The CllCl antibody alsorecognizes kininogen antigen in human platelets and neutralizes thecoagulant activity of HMWK. These combined studies indicate that theCllCl antibody is directed to the HMWK light chain. Both monoclonalantibodies produced from the cell hybrids make antibodies of thesubclass IgG₁, kappa light chain. The purified antibodies have amolecular weight on non-reduced 8% sodium dodecyl sulfate polyacrylamidegels of 200 kDa, which upon reduction yields 50 kDa and 28 kDafragments. The antibodies are conformationally specific since they donot recognize kininogen antigen that has been treated with sodiumdodecyl sulfate.

HMWK is purified from plasma by a method that relies on the differencebetween isoelectric points, and therefore differences in dissociationconstants, between HMWK and other plasma proteins. This procedure, whichconsists of quarternary ethyl aminoethyl Sephadex (QAE) chromatography,ammonium sulfate precipitation, and sulfopropyl (SP) chromatography,results in a purified protein with greater than 98% homogeneity asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Other purification methods may be used, including bindingwith immobilized monoclonal antibodies according to the presentinvention.

Mice are immunized with purified HMWK from plasma. BALB/c AnSkh mice arepreferred, although other strains may be used. The immunization scheduleand concentration of antigen administered should be such so as toproduce useful quantities of suitably primed splenocytes.

Upon completion of the immunization regimen, more fully described below,the mice are sacrificed and their spleens removed. A suspension ofsplenocytes in a suitable dium is prepared. Approximately 2.5-5 ml ofmedium per spleen is sufficient. The protocols for in vitro cellsuspension are well established.

The spleen cells are fused with mouse myeloma cells by means of a fusionpromoter. The preferred fusion promoter is polyetheylene glycol,molecular weight 1,300-1,600. Other promoters may be used. The mousemyeloma cell line is preferably one of the "drug-resistant" types, toenable selection of hybrids. The most frequently used class of myelomasare the 8-azaguanine-resistant cell lines, which are widely known andavailable. These cell lines lack the enzyme hypoxanthine guaninephosphoribosyl transferase and therefore do not survive in "HAT"(hypoxanthine-aminopterinthymidine) medium. The use of myeloma cellswith different genetic deficiencies (e.g., other enzyme deficiencies,drug sensitivities, etc.) that can be selected against in mediumsupporting the growth of genotypically competent hybrids is alsopossible. Additionally, it is suggested that the myeloma cell lineshould not itself produce any antibody, although in some circumstances,such as in the production of the CllCl antibody, secreting myeloma celllines can be used.

While the preferred fusion promoter is polyethylene glycol of averagemolecular weight 1,300-1,600 (available from ATCC), other known fusionpromoters may be used.

Fusion of cells may be carried out in an adherent monolayer, such asaccording to the method described by T. J. McKearn in "Fusion of Cellsin an Adherent Monolayer" Monoclonal Antibodies: Hybridomas: A NewDimension In Biological Analysis, (Kennett, R. H., McKearn, T. J., andBechtol, K. B., eds., Plenum Press, New York and London, 368-369, 1980).Other fusion techniques may be employed A cell ratio of 2-5:1 spleencells per myeloma cell may be used. This ratio may be varied dependingon the source of spleen or myeloma cells.

A mixture of unfused myeloma cells, unfused spleen cells and fused cellsare distributed for culturing in separate compartments (e.g., the wellsof microtiter plates) in a selective medium in which the unfused myelomacells will not survive. Distribution of the cells may be by resuspensionin a volume of diluent which is statistically calculated to isolate adesired number of cells per compartment. See, McKearn, T. J., "Cloningof Hybridoma Cell Lines by Limiting Dilution in Fluid Phase" inMonoclonal Antibodies, p. 374.

When HAT is used as the medium, unfused 8-azaguanineresistant myelomacells will not grow. Unfused spleen cells will normally die after a fewdays, since they are non-malignant. Culturing proceeds for a timesufficient to allow their death. Fused cells continue to reproduce andgrow in the selective medium.

The supernatant in each container or compartment having hybrid cellgrowth is screened and evaluated for the presence of antibody to humankininogen. Any suitable antibody-binding detection method may be used,e.g., enzymelinked immunosorbent assay, radioimmunoassay, etc.

After selection and cloning, monoclonal antibody to human kininogen maybe produced by in vitro culturing of the hybridomas or by in vivoperitoneal exudate induction in mice. The first method will yieldmonoclonal antibody of higher purity. The antibody is recovered from thesupernatant essentially free of undersired immunoglobulin. Antibodyconcentrations of 25-50 micrograms/ml are possible by this method. Ingrowth media containing serum (such as fetal calf serum) a small amountof other immunoglobulin is present.

Where concentrations of antibody larger than those obtained by in vitroculturing of hybridomas are required, the subject hybridomas may beinjected into the peritoneal cavity of syngeneic or semisyngeneic mice.After a suitable period of incubation, the hybridomas cause formation ofantibody-secreting tumors, which will produce 4-10 mg of antibody per mlof peritoneal exudate of the injected mouse. Since mice have normalantibodies in their blood and ascites, a contamination of about 5% fromthe host mouse in inevitable. Purification of ascites monoclonalantibody may remove these contaminants. The resultant antibody is ofhigh titer, being active at dilutions of 1:300,000 or higher.

The following procedure is illustrative of methods for purifying HMWKimmunogen for use in preparing hybrid cell lines according to thepresent invention.

I. Preparation Of The Immunogen

HMWK is purified according to the procedure of Kerbirious and Griffin,J. Biol. Chem. 254, 12020 (1979) as follows, with modifications. Alloperations are carried out in plastic material since glass activates thecoagulation system. All reagents are purchased from Sigma Chemical Co.0.1M epsilon aminocaproic acid (EACA) is added to all buffers.

Four hundred fifty ml of blood from two donors are freshly collectedinto 50 ml of 3.8 g % (i.e., g/dl ) sodium citrate containing 37.5 mgsoybean trypsin inhibitor (SBTI)/50 ml, 150 mg polybrene/50 ml, 10 mMEDTA, 10 mM benzamidine and 0.02% NaN₃. Platelet-poor plasma is preparedby centrifugation at 1,000 X_(g) for 30 min at 23° C.Diisopropylfluorophosphate (DFP) (2 mM final concentration) is added tothe platelet-poor plasma before the purification. (DFP is also added topooled material containing HMWK after the ammonium sulfate precipitationdescribed below and prior to chromatography on SP Sephadex). Once theplasma is incubated for 30 min with DFP, it is then diluted 2:3 with0.03 M Tris Cl, 3 mM benzamidine, 3 mM EDTA, 0.06% NaN₃ and 0.3 M EACA,pH 8.0. The plasma is then absorbed in a batch fashion on QAE Sephadex.The gel is equilibrated with 0.01 M Tris, 1 mM benzamidine, 1 mM EDTA,0.1 M EACA, pH 8.0 containing 50 microgram/ml polybrene (4-6 mohm).After the plasma is applied, the column is washed with 0.121 M Tris,0.044 M succinic acid, 0.103 M NaCl, 1 mM EDTA, 1 mM benzamidine, 0.02%NaN₃ and 0.1 M EACA, pH 7.7 (14-15 mohm) until the absorbance at 280 nmis less than 0.1. HMWK is eluted from the anion exchanger with 0.199 MTris, 0.075 M succinic acid, 0.182 M NaCl, 1 mM EDTA, 1 mM benzamidine,0.02% NaN₃ and 0.1 M EACA, pH 7.4 (23-25 mohm). After 50% ammoniumsulfate precipitation and dialysis of the resuspended precipitateagainst 0.05 M sodium acetate, 0.075 M NaCl, 1 mM EDTA, 1 mMbenzamidine, 0.02% NaN₃ and 0.1 M EACA, pH 5.3 (7-9 mohms), the HMWK isapplied to a SP Sephadex column equilibrated in the same buffer. Afterwashing the column, a linear gradient is applied with 0.1 M sodiumacetate, 0.15 M NaCl, 1 mM EDTA, 1 mM benzamidine, 0.02% NaN₃ and 0.1 MEACA, pH 5.3 (15-18 mohm) in the proximal chamber and the same buffercontaining 0.5 M NaCl (32-38 mohms) in the distal chamber. The HMWKelutes after 11/2 to 2 column volumes has passed from the gradient.

The following are two typical procedures for preparing hybrid cell linesaccording to the present invention, which is not intended to be limitedto the same.

II. Preparation Of The CllCl Monoclonal Antibody A. Immunization

Four male BALB/c AnSkh mice, 6 weeks old (Temple University, Skin andCancer Hospital) are immunized intraperitoneally with 100 micrograms ofsterile protein/mouse in complete Freund's adjuvant (week 0) and thenagain intraperitoneally with 200 micrograms of protein/mouse at weekone. At week two, a third 100 microgram injection in 0.1 ml incompleteFreund's adjuvant is given. At week three, a fourth intraperitonealinjection with 200 micrograms of antigen is given. Four days later, thespleens from two of these mice are removed aseptically and placed inpetri dishes containing 5 ml of RPMI-Versene (65 ml RPMI plus 35 mlversene stock--8.0 gm NaCl, 0.2 gm KCl, 2.1 gm Na₂ HPO₄ : 7H₂ O, 0.2 gmKH₂ PO₄, 1.0 gm EDTA in 1 liter H₂ O, pH 7.2). Using two syringes withsterile bent needles, the spleens are teased apart into a suspension.The suspension is transferred to a 15 ml sterile conical tube andallowed to sit for 1 min. After the large debris settles, thecell-suspension is transferred into another centrifuge tube. Two ml offetal calf serum is gently added to the bottom of the tube to form agradient, and the entire contents of the tube are centrifuged at 1000RPM for 8 minutes. After discarding the supernatant, the cells areresuspended in 2 ml of ice cold 0.83 M NH₄ Cl for 1 minute in order tolyse red blood cells. The cell suspension is diluted in 8 ml of 85% MEM(modified Eagle's Medium) plus 15% fetal calf serum. After makinganother 2 ml fetal calf serum gradient on the bottom of the tube, theentire contents are centrifuged at 1000 RPM for 8 minutes. Thesupernatant is removed and the cells are washed by gentle centrifugationat 1000 RPM for 8 minutes after resuspension in 10 ml RPMI-Versene.P3X63Ag8 secretor myeloma cells used in the hybridization procedure arewashed in the same way as the unlysed splenocytes.

B. Preparation of Splenocyte Feeder Layers

On the day of fusion, 1 ml of immune splenocytes from the 10 mlRPMI-Versene suspension of the splenocytes is removed to be used forfeeder layers. The cells are placed into a separate tube containing 5 mlof MEM +HAT +15% fetal calf serum.

C. Hybridization

Fusion is carried out as follows. After centrifugation of the myelomacells and splenocytes, 5 ml of RPMI-Versene is added to each tube. Thecontents of one tube are combined with the other. The ratio of spleen tomyeloma cells is 5:1. After combining the cells, they are washed twiceby centrifugation with RPMI-Versene. Fusion is performed by adding 1 mlof solution of PEG 1450 (38%)--RPMI-Versens (62%), pH 7.8 warmed to 37°.The cell pellet is resuspended by gently tapping the tube. After 1 min,1 ml of RPMI-Versene is added. After an additional 2 min wait, 2 ml ofRPMI-Versene is added. After an additional 5 min, 5 ml of DME (Dulbeco'sModified Eagle's Medium, Gilbco, Grand Island, N.Y.) +10% fetal calfserum is added. The cell suspension is then centrifuged at 800 RPM for 8min, and after the supernatant is removed, the cells are resuspended in10 ml of DME +10% fetal calf serum +1 ml RPMI-Versene. This tube, alongwith a tube containing the feeder layer cells, is incubated at 5% CO₂for 45 min. After the incubation, 0.1 ml aliquots of the hybrid cellsuspension are plated onto 24-well Linbro cluster plates with each wellcontaining 2 ml of DME +HAT. An aliquot of the feeder layer cells isalso placed into each well. The plates are incubated overnight at 37°with a 5% CO₂ humid atmosphere .

D. Selection and Growth Of Hybridoma

Following overnight incubation, half the volume of each of the wells onthe 24-well plate is removed and 1 ml of fresh DME +HAT is added. Thecells are allowed to grow without change in the medium. The hybrids werescreened daily and on day ten after hybridization the first colonygrowth is noted. Colony growth is maintained in DME +HAT +10% fetal calfserum. Hybridoma colonies with antibody reactive to the immunogen areselected and cloned by limiting dilution techniques. Cloning of thehybridomas is performed according to the procedure of McKearn, T. J."Cloning of Hybridoma Cells by Limiting Dilution and Fluid Phase" inMonoclonal Antibodies, p. 374 in 80% (DME+15% fetal calf serum +HT), 20%conditioned media. Conditioned media is prepared by growing the myelomacell line in DME+10% fetal calf serum and collecting the supernatant.

Cells from the hardiest clone were grown in culture and the supernatantwas harvested. This culture supernatant was designated CllCl. Theantibody was characterized by studies on the culture supernatant, aswell as by studies on purified antibody using (i) pure immunogen, (ii)unique plasmas, (iii) ELISA and (iv) competitive ELISA (CELISA).

III. Preparation Of The 2B5 Monoclonal Antibody A. Immunization

Four male or female BALB/c AnSkh mice, 8-10 weeks old (TempleUniversity, Skin and Cancer Hospital) are immunized subcutaneously with50 micrograms of protein/mouse in complete Freund's adjuvant (week 0)and then again subcutaneously with 50 micrograms of protein/mouse inincomplete Freund's adjuvant at week 5. Blood is removed and screened atweek 7 for antibodies to the immunogen using emzyme-linked immunosorbentassay. At week 11, 50 micrograms of immunogen/mouse in 0.15 M sodiumchloride are intraperitoneally injected. Four days later, blood isremoved from the retroorbital plexus of each mouse under lightanesthesia, and the two strongest positive mice are selected as spleendonors. The spleens of these animals are asceptically removed and placedin tissue cultured dishes (15×60 mm) containing Hank's balanced saltsolution ("HBSS", Gibco) to which 50 micrograms/ml of gentamycin or"PEN/STREP" (Gibco) are added. The latter is a mixture of penicillin andstreptomycin. The spleens are then transferred into other culture dishescontaining HBSS. The spleens are teased apart with sterile forceps andthen transferred into a centrifuge tube which is placed in ice for twominutes to allow large debris to settle. The cell suspension istransferred into another centrifuge tube and spun for 10 minutes at1,200 rpm. After discarding the supernatant, the cells are resuspendedin 5-10 ml of 0.17 M NH₄ Cl (ice cold) and placed in ice for 5 minuteswith occasional mixing in order to lyse red blood cells. The cellsuspension is gently underlaid into 10 ml of 1:1 dilution of HBSS:normalserum and centrifuged at 1,200 rpm for 10 minutes. Fetal calf serum("FCS") may be used as the normal serum. The cells are then washedthrice in DME. The number and variability of cells is then determined.

SP2/O-Ag14 myeloma cells used in the hybridization procedure are washedin the same way as the unlysed splenocytes.

B. Preparation Of Splenocyte Feeder Layers

On the day of fusion, non-immune splenocytes from the same mouse strainas immunized above are processed according to the same procedure withoutimmunization and without washing in DME. These non-immune splenocytesare used to prepare feeder layers as follows. The non-immune cells areresuspended in DME+HAT+20% FCS to a density of 2-4 ×10⁶ cells/ml. Thesecells are seeded onto 96-well plates (1-2×10⁵ cells/well) and incubatedin 5% CO₂ at 37° C. overnight as a sterility check for plating outhybrid cells.

C. Hybridization

Fusion is carried out as follows. 1.5 ml of immune splenocytes and 1.5ml of SP2/O-Ag14 are pipetted onto a Concanavalin A-coated plate. Thecell concentration of each cell type is adjusted so that the ratio ofsplenocytes to SP2/O cells is 2-3:1, with a total of 7-10×10⁷cells/plate. The plates are then incubated in 5% CO₂ at 37° C. for 45-60minutes to allow for attachment of the cells to Concanavalin A. Fusionis performed by adding 1 ml of a 50% DME:PEG solution to each plate,drop by drop. The plates are left standing for 15 seconds after theaddition of the first drop. The cells are then washed twice with 5 ml ofDME. Following addition of 5 ml of DME +20% FCS/plate, the cells areincubated overnight.

D. Selection And Growth Of Hybridomas

Following overnight incubation, the cells from the above hybridizationprocedure are transferred into centrifuge tubes and spun at 1,500 rpmfor 15 minutes. The supernatants are discarded. The cells from each tubeare suspended in 40-45 ml of DME +HAT +20% FCS and transferred into the96-well plates (0.1 ml cell/well) containing non-immune splenocytefeeder layers as prepared above. The plates are cultured with 10% CO₂ at37° C. in a humid atmosphere. The cells are allowed to grow for 3-5days, after which an additional 0.1 ml of DME +HAT +20% FCS are added toeach well. Hybrids are checked daily. Three to four weeks after fusion,the cells are switched to DME +HAT +10% FCS (no aminopterin). Hybridomacultures with antibody reactive to the immunogen are selected and clonedby a limiting dilution technique.

Cells from the strongest positive culture, designated 2B5, were injectedintraperitoneally (about 2×10⁶ cells in 0.5 ml PBS/mouse) into fourBALB/c mice which are primed 10-14 days previously with 0.5 ml ofpristane (2,6,10,14-tetramethylpentadecane). After 7-14 days, the bloodwas removed from the retro-orbital plexus of each mouse under lightether anesthesia and the tumor-induced ascites fluid was harvested. Theantibody titer of 2B5 was determined in the ascites fluid using pureimmunogen and ELISA. The antibody was characterized by studies onascites as well as by studies on purified antibody using (i) pureimmunogen, (ii) unique plasmas, (iii) ELISA, and (iv) competitive ELISA("CELISA").

IV. Purification Of CllCl and 2B5 Antibody

Antibody from culture supernatant and ascites fluid prepared above arepurified as follows. Antibody in the culture fluid is initiallyconcentrated using ammonium sulfate. One hundred ml of culture fluid issubjected to two successive 50% ammonium sulfate precipitationsfollowing by overnight dialysis against 0.01 M Tris Cl, 0.15 M NaCl, pH7.4 (TBS). After dialysis, the material is concentrated to 1 to 2 ml.Concentrated culture supernatant antibody or ascites is then diluted 1:1in the binding buffer for a modified Protein A affinity chromatographysystem (Affi-Gel Protein A, Bio Rad Corp., Richmond, Calif.). Afterslowly applying the crude material to the Protein A column and thenwashing the column with binding buffer such that the absorbance at 280nm is less than 0.025, the antibody is eluted with an acidic buffer. Thepeak of elution is pooled and immediately dialysed against TBS. Finalantibody is concentrated to 2 to 5 mg/ml. The quality of thepurification is monitored by determining the residual amount of antibodythat does not bind to the column or does not elute from the column byperforming direct binding ELISA on the chromatography wash andregeneration material. The purity of the final antibody preparation isdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisaccording to the procedure of Laemmli, Nature 227, 680 (1970).

V. Characterization Of The Monoclonal Antibodies

The two monoclonal antibodies of the present invention reacted withbiochemically pure plasma HMWK by direct-binding ELISA. Studies wereinstituted to determine which portion(s) of the HMWK molecule theseantibodies were directed toward.

Initial studies were performed to determine whether either of themonoclonal antibodies neutralized HMWK coagulant activity. Each purifiedantibody was first tested to determine if it had any coagulant activityitself. Neither purified antibody had any intrinsic coagulant activity.Normal plasma was diluted and then incubated with decreasing dilutionsof purified monoclonal antibodies (from 1:10 to 1:2500) overnight at 4°.The next day the ability of this antibody-treated normal plasma tocorrect the coagulant activity of total kininogen-deficient plasma wastested (FIG. 1). As can be seen in the figure, the CllCl antibody at thelower dilution completely inhibited the coagulant activity of normalplasma whereas the 2B5 antibody did not. These findings suggest that theCllCl antibody is directed to the light chain of HMWK--that portion ofthe molecule which contains the coagulant portion--or to a site on theheavy chain adjacent to the light chain which can sterically interferewith the latter's function. The 2B5 antibody, which did not neutralizethe coagulant function of the HMWK molecule, must be directed to theheavy chain or a portion on the light chain of HMWK not involved withcoagulant function. In order to clarify what segment of the kininogenmolecule these monoclonal antibodies were directed toward, variousimmunochemical studies were performed.

Initial immunochemical studies utilized a competitive enzyme-linkedimmunochemical assay (CELISA) developed by Schmaier et al, J. Clin.Invest. 71, 1477 (1983). Since nature has provided genetic variantplasmas which have either an absence of HMWK or total kininogen (bothhigh and low molecular weight kininogen) the ability of each of theseplasmas to consume the monoclonal antibodies was tested. As shown inFIG. 2, both normal plasma and HMWK-deficient plasma (FitzgeraldPlasma), but not total kininogen-deficient plasma (Williams Plasma),decreased the titer of the 2B5 antibody. This finding suggests that the2B5 antibody is directed to the portion of the HMWK molecule common toboth normal and HMWK-deficient plasma. Since the latter plasma contains40% normal LMWK levels, and since the LMWK heavy chain is identical tothe HMWK heavy chain, the 2B5 antibody must be directed to the HMWKheavy chain.

The specificity of the 2B5 antibody for the HMWK heavy chain was furthertested by immunoblot studies (FIG. 3). Initial studies using a directbinding ELISA and immunoblot showed that the 2B5 antibody was aconformationally specific antibody since it did not recognize HMWKantigen that had been treated with sodium dodecyl sulfate. An 8%alkaline polyacrylamide gel electrophoresis system was used to performthe immunoblot studies (Davis, B. J., Ann. N.Y. Acad. Sci. 121, 404,1964). Purified HMWK, HMWK-deficient plasma, and normal plasma wereapplied to the alkaline gel and electrobloted onto nitrocellulose(Towbin, et al, Proc. Natl. Acad. Sci. 76, 1350, 1979). An immunoblotwas performed using the 2B5 antibody as the primary antibody and ¹²⁵I-goat anti-mouse affinity-purified antibody as the second antibody. Ascan be seen in FIG. 3, the 2B5 antibody recognizes kininogen antigenfrom normal plasma as two bands and kininogen antigen in HMWK-deficientplasma as one band. This finding indicates that the 2B5 antibody isdirected to the heavy chain of HMWK, which is identical to the heavychain of LMWK.

The affinity of the 2B5 antibody for the HMWK heavy chain was studiedfurther. Data from the competition inhibition of the 2B5 antibody bynormal plasma was applied to a computer program to fit the curve by afour-parameter logistic function (Cannellas, P. F., Karu, A. E., J.Immunol. Meth. 47, 375, 1981) in order to determine the calculatedmidpoint of the curve (FIG. 4). This plot is also a standard curve for aquantitative CELISA. Using the calculated midpoint of the competitioninhibition curve, the affinity of the antibody for antigen wasdetermined by the formula of Muller (Meth. Enzymol. 92, 589, 1983), Ka=8/3(<I_(t) >-<T_(t) >). The calculated apparent K_(d) of the antibodyfor antigen was 1.91 nM. This finding was confirmed by direct bindingstudies as follows.

Purified 2B5 antibody was radiolabeled with ¹²⁵ I by the Iodogentechnique (Fraker, P. J., Speck, S. C. Biochem. Biophys. Res. Commun.80, 849, 1978). Purified HMWK was linked to polystyrene cuvette wells.The radiolabeled antibody in the absence or presence of a fifty-foldmolar excess of unlabeled antibody was incubated in the antigencoatedcuvette wells. After subtracting non-specific binding from the Bound andFree portions, the slope of the line of Bound over Free versus Bounddetermines the affinity constant of the antibody for its antigen.Performing this study with the 2B5 antibody, the calculated apparentK_(d) of this antibody for the heavy chain of the kininogen moleculeswas 1.76 nM.

Final characterization of the 2B5 antibody was performed by affinitychromatography (FIG. 5). Purified 2B5 antibody was linked to cyanogenbromide (CNBr)-activated Sepharose 4B (Meth. Enzymol. 82, 87, 1980).Purified HMWK was cleaved by purified plasma kallikrein (ratiokallikrein: kininogen =1:130). The cleavage fragments were alkylatedwith iodoacetamide and reduced with dithiothreitol and applied to the2B5 affinity column. As can be seen in FIG. 5, the two HMWK light chains(56 kDa, 46 kDa) did not bind to the column. However, the 64 kDa heavychain did bind and had to be eluted with 4 M guanidine. On furthertesting of this affinity-purified material, only the unbound portion ofthe molecule contained the coagulant activity of HMWK. These combinedstudies indicate that the 2B5 antibody is directed to the heavy chain ofthe human plasma kininogen molecules.

Immunochemical studies were also performed on the CllCl antibody. UsingCELISA, the CllCl antibody was consumed by kininogen antigen in normalplasma but not by kininogen antigen in HMWK-deficient plasma (FIG. 6).Not shown in the figure, total kininogen-deficient plasma did notconsume the CllCl antibody. This finding suggests that the CllClantibody is directed to the HMWK light chain since the only differencebetween the two plasmas was the presence of the light chain of the HMWKmolecule in normal plasma.

The specificity of the CllCl antibody for the HMWK light chain wasfurther tested on immunoblot studies. The antibody is similar to the 2B5antibody in that it does not recognize antigen whose conformation mayhave been changed by the presence of sodium dodecyl sulfate. The samealkaline polyacrylamide gel electrophoresis, electroblotting andimmunoblotting techniques as used for the 2B5 antibody (FIG. 3) werecarried out with the CllCl antibody using normal plasma andHMWK-deficient plasma as a source of antigen. As can be seen in FIG. 7,the CllCl antibody recognized kininogen antigen in normal plasma but notkininogen antigen from HMWK-deficient plasma. This study confirms thatthe CllCl antibody is directed to the HMWK light chain.

The affinity of the CllCl antibody for HMWK was studied by the sametechniques employed for measuring the binding of the 2B5 antibody.Plotting the competition inhibition of the CllCl antibody by normalplasma on a fourparameter logistic function to determine its calculatedmidpoint of inhibition resulted in a calculated affinity of CllCl forits antigen of about K_(d) =0.78 nM (FIG. 8). This plot is also astandard curve for a quantitative CELISA.

VI. Functional Characteristics Of The Monoclonal Antibodies

Although the CllCl antibody neutralized the coagulant activity of HMWK,further studies were performed to determine if the 2B5 antibodyinfluenced the activation of HMWK coagulant activity. Normal plasma wasincubated with the 2B5 antibody overnight at 4°. Aliquots of the2B5-treated normal plasma were compared with normal plasma alone todetermine if it altered the activation of the HMWK coagulant activity.Normal plasma had peak HMWK activity when incubated with HMWK activatingsurface and phospholipid for nine minutes. 2B5-treated normal plasmaexhibited a similar rate and extent of activation as normal plasmaalone. This negative study indicates that the 2B5 molecule is directedto a portion on the HMWK heavy chain that in no way influences HMWKcoagulant activity.

Further studies were performed to determine if 2B5 or CllCl recognizeplatelet high molecular weight kininogen. Using an immunoprecipitationtechnique with protein A, the CllCl antibody immunoprecipitated a formof platelet high molecular weight kininogen which was similar in size(120 kDa) to plasma and purified plasma high molecular weight kininogen(FIG. 9).

Additional studies were performed to determine if either the 2B5 orCllCl monoclonal antibody influenced the ability of the HMWK molecule tobind to its platelet receptor. In separate but simultaneous experimentsthe 2B5 or CllCl antibody independently almost completely inhibitedexogenous ¹²⁵ I-HMWK binding to unstimulated platelets. This findingsuggested that the HMWK molecule has two sites which are required forits association with the platelet surface.

VI. Quantitative Assay

The monoclonal antibodies of the present invention are useful indetecting changes in HMWK and LMWK concentrations in human plasma. Theantibodies may be used to assay unknown samples of plasma for HMWK andtotal kininogen in a quantitative fashion by the following techniquedeveloped in our laboratory using a polyclonal antibody, Schmaier et al,J. Clin. Invest. 71, 1477 (1983).

A. Quantitative Assay Of HMWK With CllCl Antibody

Plasma concentrations of HMWK are determined by CELISA as follows. TheCllCl monoclonal antibodies comprise the primary antisera and rabbitanti-mouse antibody conjugated with alkaline phosphatase is used as thesecondary antibody. On day 1 100 ng of purified HMWK in 0.1 M Na₂ CO₃ pH9.6 is linked to the surface of polystrene cuvette wells (GilfordInstrument Laboratories Inc., Oberline, Ohio) by overnight incubation at37° C. On the same day, incubation mixtures, in 1.5 ml conicalpolypropylene tubes (No. 72.690, Sarstedt, Inc., Princeton, N.J.)precoated with 0.2% bovine serum albumin, are made containing thefollowing: 0.2 ml of antigen (purified HMWK or plasma solubilizedplatelets) diluted in 0.001 M sodium phosphate pH 7.4, 0.15 M NaClcontaining 0.05% Tween-20 (PBS-Tween) and 0.2 ml of a 1:2500 dilution ofCllCl antibody . These samples are then incubated overnight at 37° C. Onday 2 each antigen-linked cuvette well is washed three times withPBS-Tween and incubated for 1 h at 37° C. with 0.2% radioimmunoassaygrade bovine serum albumin. After washing, 0.2 ml from each incubationmixture is added to each cuvette well and incubated for 2 h at 37° C. Atthe conclusion of this incubation the rewashed wells are exposed to 0.2ml of a 1:500 dilution in PBS-Tween of rabbit anti-mouse antibodyconjugated with alkaline phosphatase. After another 2.5 h incubation at37° C., the washed cuvettes receive sequentially timed additions of 0.4ml of p-nitrophenylphosphate disodium (1 mg/ml) in 0.05 M Na₂ CO₃, 1 mMMgCl, pH 9.8. At precise time intervals (10-20 min) after the additionof the substrate to each well, the amount of hydrolysis of the substratein each well is either stopped with sequential additions of 0.4 ml of 2M NaOH (final concentration 1 M NaOH) or was sequentially measuredspectrophotometrically in a PR50 EIA Processor-Reader (GilfordInstrument Laboratories, Inc.) at 405 nm.

The CELISA assay is standardized against purified HMWK. Purifiedsingle-band HMWK is diluted according to its protein concentrationdetermined by protein assays. Protein assay may be according to themethod of Lowry et al., J. Biol. Chem. 193, 265-275 (1951) or Bradford,Anal. Biochem. 72, 248 (1976), for example. The amount of HMWK antigenin one pool of normal human plasma (lot No. NIO George King, Biomedical,Inc.) is compared with the purified HMWK diluted in totalkininogen-deficient plasma on electroimmunodifusion using polyclonalanti-HMWK antisera. All subsequent dilutions of pooled normal plasma arebased on the amount of HMWK antigen determined in the pool. Samples forthe standard curve (purified HMWK, plasma) and unknowns (plasma) areboth assayed at 10 different dilutions in triplicate. All data areanalyzed on a TRS-80 model III computer (Tandy Corp., Forth Worth,Tex.). Raw optical density readings are averaged after the blanks aresubstracted. Outliers are defined as points with a greater than 10% SD.A best-fit standard curve is determined by nonlinear regression. Thebest-fit standard curve is then entered into an iterative computerprogram that uses a four-parameter logistic function and an optionalweighting function for a sigmoidal line fit of the standard curve.Canellas, et al, J. Immunol. Methods 47, 375-385 (1981). The weightingfunction is determined by a multiple linear regression for each batch ofantisera. Id. This program yields a semilogarithmic graph (FIG. 8) inwhich the ordinate is the optical absorbance calculated in relativevalues from 0 to 1 and the abscissa is the absolute amount of antigenincubated with antisera. Unknown plasma samples are determined by linearleast squares regression from the standard curve of the iterativeprogram and the calculated slope of the regression line is an estimateof the specific activity of the sample. Test sample determinations thatare considered valid are computed results that fall on the linearportion of the standard curve for the individual assay and arecharacterized by a regression analysis having p values less than 0.05 inan F test on the determination of the line and in a t test against thenull hypothesis that the slop of the line equals zero. The CllClantibody may thus be used to quantitatively assay unknown samples forHMWK content.

B. Quantitative Assay of Total Kininogen/LMWK With 2B5 Antibody.

The above assay procedure, repeated with the 2B5 antibody, provides aquantitative assay for total kininogen content. The data, when analyzedand plotted in accordance with the above-described four-parameterlogistic function, results in the curve shown in FIG. 4, which may beused to assay unknown samples for total kininogen content. Subtractingthe HMWK content of a plasma as measured by the CllCl antibody from theplasma's total kininogen content as measured by the 2B5 antibodyindicates the LMWK concentration in the plasma.

The antibodies may thus be used to diagnose activation of the contactcoagulation system, which can be signalled by changes in the levels ofthe plasma kininogens.

Detection and quantitation of kininogen in any specimen of interest canbe carried out with the present monoclonal antibodies using a CELISA ora radioimmunoassay. Techniques have already been developed to radiolabelthese antibodies. As indicated in the CELISA experiments which led toFIGS. 2, 4, 6 and 8, the antibodies are incubated with the specimen, andthe material bound thereto is measured indirectly by the amount ofenzyme-absorbed secondary antibody bound to a cuvette well. Otherquantitative immunological assays may be used, including, but notlimited to CELISA, radioimmunoassay, ELISA, fluorescent-antibody assay,precipitation, agglutination, and electroimmunodiffusion. Such methodsalso include fast liquid chromatographic methods such as ion exchange,gel filtration and reverse phase chromatography.

The present monoclonal antibodies are thus useful to measure kininogenlevels in the plasma in pathogenic states related to the contactactivation of the coagulation pathway. These pathogenic states include,but are not limited to, acquired disorders such as gram negative orpositive sepsis, peridontal disease, carcinoid syndrome, postgastrectomysyndrome, nephrotic syndrome, type IIa hyperlipoproteinemia, allograftrejection, allergic reactions, typhoid fever, viremia, and RockyMountain Spotted Fever. The monoclonal antibodies are also useful inmeasuring abnormal kininogen levels connected with hereditary disorderssuch as hereditary angioedema.

Apart from plasma, other specimens of interest include saliva, nasalsecretions, platelets, kidney, urine, leukocytes, skin, heart, liver andendothelial cells.

Since the CllCl antibody is specific for HMWK and does not bind LMWK,while the 2B5 antibody binds both LMWK and HMWK, the antibodies may beused in serial fashion to determine the level of HMWK and LMWK in thesame sample.

The monoclonal antibodies of the present invention are also useful inremoving high and low molecular weight HMWK and LMWK from the blood orany body fluid or suspension, thereby providing a method of therapy forany possible (but not yet demonstrated) disease condition wherein thepatient may suffer from increased levels of these proteins. Followingremoval of excessive amounts of these proteins from blood, the tissuemay be returned to the body.

A method for purifying human plasma kininogen is provided. Themonoclonal antibodies of the present invention may be easily covalentlybound to an immobilized matrix such as CNBr-activated Sepharose, asdisclosed above. Both plasma kininogens may be purified by passingplasma through such a column containing immobilized monoclonal antibody,and then eluting the bound kininogen. Purification of the kininogens inthis manner may take place with or without prior partial purification,such as by ion exchange chromatography.

A column of the type described above may also be used to separate thelight and heavy HMWK chains since the two antibodies of the presentinvention are specific for different epitopes on the light and heavychains. Moreover, the heavy chain-directed antibody 2B5 can be used topurify LMWK directly from plasma. Finally, because HMWK bindsprekallikrein and factor XI, columns of this type charged with HMWK maybe used to purify these proteins.

The hybridomas of the present invention secrete antibody belonging tosub-class IgG₁, which means the antibodies have the same "constant"region. An antibody to a specific antigen has a constant region and a"variable" The variable region recognizes antigen, regardless of thetype of constant region. Thus, monoclonal antibodies exhibiting thecharacteristics described herein may be of sub-class IgG₁, IgG_(2a),IgG₃, IgM, IgA or other Ig classes. Since the difference inimmunoglobulin class (Ig) will not effect the pattern of reactivity ofthe antibody toward the antigen, it is contemplated that all monoclonalantibodies to human kininogen are included within the subject inventionregardless of Ig class or sub-class.

The method of the present invention for preparing monoclonal antibodiesto human kininogen, which includes immunization, fusion and selection ofhybridomas, may be followed to generate cell lines other than the twocell lines disclosed herein. Because individual hybridomas may beidentified only by the antibody which they produce, it is contemplatedthat any hybridoma-producing antibody to any human kininogen is includedwithin the scope of the present invention, as are methods for makingsuch antibodies employing hybridomas. It is further contemplated thatsplenocytes and myelomas from other vertebrates, such as human, rat,bovine, porcine, etc., not just those of murine origin, may be used toform hybridomas using the methods described herein.

The monoclonal antibodies of the present invention are produced byhybridomas. However, it is contemplated that other methods ofcell-immortalization may be used to produce monoclonal antibodiesagainst human kininogen. These methods are known to those skilled in theart. For example, human antibody-producing lymphocytes may beimmortalized by transformation with Epstein-Barr virus. See, e.g.,Chiorazzi, et al, J. Exp. Med. 56, 930-35 (1982); Steintz, et al, J.Immunol. 132, 877-82 (1984).

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan the foregoing specification, as indicating the scope of theinvention.

What is claimed:
 1. A method for determining the level of humankininogen in a specimen comprising contacting the specimen with amonoclonal antibody which binds to an antigenic determinant of humankininogen, and measuring the human kininogen bound by said antibody byan assay means, which antigenic determinant is characterized by bindingto monoclonal antibody produced by hybridoma ATCC #HB-8963 or ATCC#HB-8964.
 2. A method according to claim 1 wherein the specimen isselected from the group consisting of plasma, saliva, nasal secretions,platelets, kidney, urine, skin, leukocytes, heart, liver, andendothelial cell.
 3. A diagnostic method according to claim 2 whereinthe monoclonal antibody is produced by a hybridoma selected from thegroup of the hybridomas numbered ATCC #HB-8963 and ATCC #HB-8964.
 4. Adiagnostic method according to claim 1 wherein said assay means is animmunological assay means selected from the group consisting ofradioimmunoassay, enzyme-linked immunosorbent assay, CELISA, fluorescentassay, radial immunodiffusion, precioitation, electroimmunodiffusion,and agglutination.
 5. A diagnostic method according to claim 1 whereinthe assay means is fast liquid chromatography.